Reports arose from major Chinese wheat production regions that flixweed was not controlled by tribenuron after the herbicide was continuously used for several years. Flixweed seeds were collected from wheat fields that had been treated with tribenuron repeatedly over 3 to 15 yr or from road sides and remote hills that had never received tribenuron in Jiangsu, Hebei, Shanxi, Sichuan, Shandong, Shaanxi, and Henan provinces, and Tianjin and Beijing metropolises in China. The response of various biotypes to tribenuron was determined by whole plant experiments in the greenhouse. The experiments demonstrated that 11 of 32 flixweed biotypes were susceptible to tribenuron. The remaining 21 biotypes expressed moderate to high levels of tribenuron resistance with resistance indices ranging from 4 to > 1,500. DNA sequence analysis of acetolactate synthase (ALS) genes of selected biotypes 6, 7, 27, and 29 revealed a point mutation at position 197 of the ALS gene, as numbered relative to the protein sequence of Arabidopsis, where proline was substituted by leucine in biotype 7 and by threonine in biotype 29. These mutations are known to confer resistance to ALS-inhibiting herbicides and are responsible for the high resistance of these biotypes to tribenuron. The results also indicate that tribenuron resistance in flixweed is widespread in China and management programs to control these resistant populations are warranted.